Revisiting plant stress memory: mechanisms and contribution to stress adaptation.

Highly repetitive adverse environmental conditions are encountered by plants multiple times during their lifecycle. These repetitive encounters with stresses provide plants an opportunity to remember and recall the experiences of past stress-associated responses, resulting in better adaptation towards those stresses. In general, this phenomenon is known as plant stress memory. According to our current understanding, epigenetic mechanisms play a major role in plants stress memory through DNA methylation, histone, and chromatin remodeling, and modulating non-coding RNAs. In addition, transcriptional, hormonal, and metabolic-based regulations of stress memory establishment also exist for various biotic and abiotic stresses. Plant memory can also be generated by priming the plants using various stressors that improve plants' tolerance towards unfavorable conditions. Additionally, the application of priming agents has been demonstrated to successfully establish stress memory. However, the interconnection of all aspects of the underlying mechanisms of plant stress memory is not yet fully understood, which limits their proper utilization to improve the stress adaptations in plants. This review summarizes the recent understanding of plant stress memory and its potential applications in improving plant tolerance towards biotic and abiotic stresses.

California TRV-based VIGS vectors mediate gene silencing at elevated temperatures but with greater growth stunting.

BACKGROUND: Tobacco rattle virus (TRV) based virus-induced gene silencing (VIGS), a widely used functional genomics tool, requires growth temperatures typically lower than those of the plant's native environment. Enabling VIGS under native conditions in the field according to applicable safety regulations could be a revolutionary advance for ecological research. RESULTS: Here, we report the development of an enhanced thermal tolerant VIGS vector system based on a TRV California isolate. cDNA clones representing the whole viral genome were sequenced and used to construct separate binary plant transformation vectors for functional elements of RNA1 (6765 nt) and RNA2 (3682 nt). VIGS of target genes was induced by transient transformation of the host plant with both vectors or by treating the host plant with sap from already VIGS induced plants. In Nicotiana attenuata the silencing efficiency of the PDS (phytoene desaturase) gene was 90% at 28 °C and 78% at 30 °C. Silencing at these temperatures was more prominent and durable than silencing induced by the widely used TRV PpK20-based pBINTRA6/pTV00 system, but was associated with a viral phenotype. Differences in the suppressor protein and RNA dependent RNA polymerase sequences between the TRV California isolate and PpK20 may be the reason for their different thermal tolerance. CONCLUSIONS: The new TRV California-based VIGS vectors induce gene silencing in Nicotiana attenuata at higher temperatures than the existing pBINTRA6/pTV00 vector system, but cause greater growth defects. The new vector system opens up an avenue to study genes functions in planta under field conditions.

Improvement in yield attributes and fatty acids composition in the derivative hybrids compared to their respective parents in Indian mustard (Brassica juncea L.).

Erucic acid, more than 2 %, in mustard seed oil is considered unhealthy as edible oil, and also anti-nutritional for human consumption. The existing mustard varieties of Bangladesh contain 40-48 % erucic acid, which is a big concern for the country's nutritional, and food security and safety. Hence, to improve the seed oil quality of the existing variety, six popular cultivars of Brassica juncea mustard were crossed with a canola-grade line in 7 × 7 half diallel fashion, and the developed 21 F1 hybrids were assessed for yield contributing traits, and fatty acids composition. Variables with significant variations were found, while days to siliquae maturity, plant height, days to first flowering, and seeds per siliquae have moderate narrow sense heritability. The estimated gene action indicated that dominant or over-dominant gene action was more prominent in governing the traits. The parents, P1, P3, and P4 were discovered the best general combiners for early maturity and short phenology, whereas P2 and P7 were found to be the best general combiners for yield-attributing traits. Moreover, the hybrids P1 × P4, P1 × P6, P2 × P7, P4 × P6 and P3 × P5 were chosen as the promising hybrids due to their best specific combining ability, and desired heterotic effects on yield contributing traits. In addition, a significant decrease, on average 30-40 %, in erucic acid, but an approximately 20-25 % increase of oleic acid was found among the hybrids, in which the hybrids P1 × P6-S1, P5 × P6-S2 and P5 × P6-S4 demonstrated a better stability index. Overall, the obtained findings suggested that the hybrids, viz. P1 × P5, P1 × P6, P2 × P3, P2 × P7, P4 × P6, P5 × P6, and P6 × P7 were promising based on their early maturity, high-yielding, reduced erucic acid, and high oleic acid contents.

Mixed miniature postsynaptic currents resulting from co-release of glycine and GABA recorded from glycinergic neurons in the neonatal respiratory network.

Inhibitory neurons are involved in the generation and patterning of the respiratory rhythm in the adult animal. However, the role of glycinergic neurons in the respiratory rhythm in the developing network is still not understood. Although the complete loss of glycinergic transmission in vivo is lethal, the blockade of glycinergic transmission in slices of the medulla has little effect on pre-Bötzinger complex network activity. As 50% of the respiratory rhythmic neurons in this slice preparation are glycinergic, they have to be considered as integrated parts of the network. We aimed to investigate whether glycinergic neurons receive mixed miniature inhibitory postsynaptic currents (mIPSCs) that result from co-release of GABA and glycine. Quantification of mixed mIPSCs by the use of different objective detection methods resulted in a wide range of results. Therefore, we generated traces of mIPSCs with a known distribution of mixed mIPSCs and mono-transmitter-induced mIPSCs, and tested the detection methods on the simulated data. We found that analysis paradigms, which are based on fitting the sum of two mIPSC templates, to be most acceptable. On the basis of these protocols, 20-40% of all mIPSCs recorded from respiratory glycinergic neurons are mixed mIPSCs that result from co-release of GABA and glycine. Furthermore, single-cell reverse transcriptase polymerase chain reaction revealed that 46% of glycinergic neurons co-express mRNA of glycine transporter 2 together with at least one marker protein of GABAergic neurons. Our data suggest that significant co-transmission occurs in the pre-Bötzinger complex that might be involved in the shaping of synaptic inhibition of respiratory glycinergic neurons.

Persistent activity in layer 5 pyramidal neurons following cholinergic activation of mouse primary cortices.

Persistent spiking activity is thought to be a cellular process involved in working memory. We have been interested in whether persistent activity also exists in cortical areas which are not involved in this memory process. To study the possible presence and the mechanisms of persistent activity in layer 5 pyramidal cells of the mouse primary somatosensory, visual and motor cortices, we used patch-clamp and calcium imaging techniques. A combination of cholinergic receptor activation and suprathreshold depolarization or sufficient extracellular stimulation leads to either a subthreshold afterdepolarization or suprathreshold persistent activity in these cortices. There is a continuum of response amplitudes depending on depolarization size. To initiate persistent activity, spikes have to be induced at a frequency of at least 20 Hz, if tested for 1 s. Acetylcholine muscarinic, but not nicotinic, receptors are important for initiating persistent activity. Persistent activity is an intrinsic cellular, not a network, phenomenon as it persists under blockade of ionotropic glutamate and GABA receptors. A rise in intracellular calcium concentration through voltage-gated calcium channels is needed for persistent activity initiation, while intracellular calcium stores are not crucial. The increased intracellular calcium concentration leads to the activation of calcium-sensitive nonspecific cationic channels. This study for the first time describes the presence and the underlying mechanisms of persistent activity in pyramidal cells of three primary sensory and motor cortex areas. These results thereby suggest that persistent activity may be a general capability of deep layer cortical pyramidal cells.

Genetic ablation of VIAAT in glycinergic neurons causes a severe respiratory phenotype and perinatal death.

Both glycinergic and GABAergic neurons require the vesicular inhibitory amino acid transporter (VIAAT) for synaptic vesicle filling. Presynaptic GABA concentrations are determined by the GABA-synthesizing enzymes glutamate decarboxylase (GAD)65 and GAD67, whereas the presynaptic glycine content depends on the plasma membrane glycine transporter 2 (GlyT2). Although severely impaired, glycinergic transmission is not completely absent in GlyT2-knockout mice, suggesting that other routes of glycine uptake or de novo synthesis of glycine exist in presynaptic terminals. To investigate the consequences of a complete loss of glycinergic transmission, we generated a mouse line with a conditional ablation of VIAAT in glycinergic neurons by crossing mice with loxP-flanked VIAAT alleles with a GlyT2-Cre transgenic mouse line. Interestingly, conditional VIAAT knockout (VIAAT cKO) mice were not viable at birth. In addition to the dominant respiratory failure, VIAAT cKO showed an umbilical hernia and a cleft palate. Immunohistochemistry revealed an almost complete depletion of VIAAT in the brainstem. Electrophysiology revealed the absence of both spontaneous glycinergic and GABAergic inhibitory postsynaptic currents from hypoglossal motoneurons. Our results demonstrate that the deletion of VIAAT in GlyT2-Cre expressing neurons also strongly affects GABAergic transmission and suggest a large overlap of the glycinergic and the GABAergic neuron population during early development in the caudal parts of the brain.

Mungbean yellow mosaic Indian virus encoded AC2 protein suppresses RNA silencing by inhibiting Arabidopsis RDR6 and AGO1 activities.

RNA silencing refers to a conserved RNA-directed gene regulatory mechanism in a wide range of eukaryotes. It plays an important role in many processes including growth, development, genome stability, and antiviral defense in the plants. Geminivirus encoded AC2 is identified as an RNA silencing suppressor protein, however, the mechanism of action has not been characterized. In this paper, we elucidate another mechanism of AC2-mediated suppression activity of Mungbean Yellow Mosaic India Virus (MYMIV). The AC2 protein, unlike many other suppressors, does not bind to siRNA or dsRNA species and its suppression activity is mediated through interaction with key components of the RNA silencing pathway, viz., RDR6 and AGO1. AC2 interaction inhibits the RDR6 activity, an essential component of siRNA and tasi-RNA biogenesis and AGO1, the major slicing factor of RISC. Thus the study identifies dual sites of MYMIV-AC2 interference and probably accounts for its strong RNA silencing suppression activity.

MYMIV-AC2, a geminiviral RNAi suppressor protein, has potential to increase the transgene expression.

Gene silencing is one of the limiting factors for transgene expression in plants. But the plant viruses have learnt to suppress gene silencing by encoding the protein(s), called RNA silencing suppressor(s) (RSS). Hence, these proteins could be used to overcome the limitation for transgene expression. The RNAi suppressors, namely HC-Pro and P19, have been shown to enhance the transgene expression but other RSS proteins have not been screened for similar role. Moreover, none of RSSs from the DNA viruses are known for enhancing the expression of transgenes. The Mungbean Yellow Mosaic India Virus (MYMIV) belonging to the genus Begomovirus within the family of Geminiviridae encodes an RSS called the AC2 protein. Here, we used AC2 to elevate the expression of the transgenes. Upon introduction of MYMIV-AC2 in the silenced GFP transgenic tobacco lines, by either genetic hybridisation or transgenesis, the GFP expression was enhanced several fold in F1 and T0 lines. The GFP-siRNA levels were much reduced in F1 and T0 lines compared with those of the initial parental silenced lines. The enhanced GFP expression was also observed at the cellular level. This approach was also successful in enhancing the expression of another transgene, namely topoisomeraseII.